MarA, SoxS and Rob (the latter two approximately 45% identical to MarA) are members of an AraC sub-family of transcriptional activators and mediate resistance to superoxides, organic solvents and a broad range of antibiotics in a number of gram-negative bacteria. The binding site or 'marbox' to which MarA, SoxS and Rob bind is located in the region immediately upstream of the binding signals (-10 and -35 sites) for RNA polymerase (RNP) and is a highly degenerate, asymmetric sequence oriented in those promoters in which it overlaps the -35 site (Class II promoters), so that the COOH-terminus of MarA lies adjacent to RNP and in the opposite orientation in those promoters where the marbox lies 15 (or 26) bp upstream of the -35 site (Class I promoters). Estimates of the frequency with which these sites appear on the genome (~10,000) and the paucity of MarA or SoxS molecules made during induction (<2000) led us to hypothesize that the activators did not first bind to the promoter site and then recruit RNP (as had been previously assumed), but rather that the activators first interacted with RNP and then scanned the chromosome for those positions having sites (-10 RNP, -35 RNP and activator binding signals) appropriately spaced (Martin, Gillette, Martin & Rosner, Molecular Microbiology, 2001, in press). The RNP-MarA complex observed had an equilibrium binding constant of ~0.3 ?M. This promises to be an entirely new paradigm for transcriptional activation. To learn more about the interactions between MarA and the highly degenerate DNA sequence at the atomic level, extensive NMR analyses have been carried out between MarA and oligonucleotides corresponding to several of the chromosomal binding sites. Among the more important observations was the demonstration that MarA interacts with DNA at three points rather than just the two observed by x-ray crystallography (Dangi, Pelupessey, Martin, Rosner, Louis and Gronenborn, J. Mol. Biol. 2001 in press). Although much had been known concerning the regulation of MarA and SoxS, Rob appeared to be unregulated. It is present in E. coli to the extent of ~10,000 molecules per cell, and though inefficient as a transcriptional activator in vivo it appears to be as effective as MarA and SoxS in vitro. We have now demonstrated that Rob can be activated by treating cells with dipyridyl. This activation requires a COOH-terminal domain of Rob (absent in MarA and SoxS) though the precise mechanism of the activation remains to be elucidated (Rosner, Dangi, Gronenborn and Martin, submitted for publication). Microarray analysis demonstrated there are >160 genes activated and >130 genes repressed when cells were treated with agents that induce MarA or SoxS. We have used techniques of informatics to predict which of these genes are directly under the control of MarA and SoxS and are currently evaluating those predictions.